DESCRIPTION (Verbatim from the applicant's abstract) Preservation injury and/or immune- mediated vascular damage result in varying levels of acute or chronic graft dysfunction. Injured vascular endothelial cells and activated platelets release substantive amounts of nucleotides that interact with multiple purinergic/pyrimidinergic type-2- (P2)-receptors expressed by vascular cells platelets and leukocytes. Extracellular nucleotides therefore could mediate signaling events that influence blood flow, hemostatic and inflammatory reactions, pertinent to both vascular injury and graft rejection. Ectonucleotidases are extracellular nucleotide-metabolizing enzymes that modulate P2-receptor-mediated signaling e.g. by catalyzing the hydrolysis of ATP and/or ADP. We have shown CD39, a B-lymphocyte activation marker, to be the prototype member of an ectonucleotidase family, now termed nucleoside triphosphate diphosphohydrolases (CD39/ NTPDases). CD39 with at least two other CD39-like(L)/ NTPDases and several defined P2-receptors (e.g. P2Yl, P2Y2, and P2X7) are expressed by quiescent endothelium. This arrangement may "fine-tune" vascular responses to fluctuating levels of these ubiquitous nucleotide messengers. In addition, NTPDase activity of CD39 is regulated at both transcriptional and post-translational levels in vitro. Importantly, vascular NTPDase activity is rapidly lost with exposure to oxidant stress in vivo e.g. in reperfusion injury and graft rejection. Conversely, over-expression of CD39 or administration of soluble NTPDases have major beneficial effects on platelet sequestration and thrombosis in several models of vascular injury tested to date. We have recently targeted and deleted cd39 by homologous recombination. The mice generated were viable but exhibited major thromboregulatory and hemostatic disturbances, ascribed to aberrant nucleotide-mediated signaling and receptor desensitization. These cd39-null mice respond adversely to ischemiareperfusion injury and their cardiac xenografts are more subject to vascular thrombosis. Deletion of cd39 also appears to result in defective monocyte and Iymphocyte responses, relevant to graft rejection responses. In this proposal, we will investigate how CD39 regulates several P2-receptor mediated events in vitro and consequent inflammatory reactions in vivo. We will examine the distribution, structural and functional interactions of CD39 with related NTPDases and those P2-receptors co-expressed in the vasculature. Pathological, immunological, molecular and biochemical studies will involve analysis of clinical biopsy material, mutant or transgenic mice and in vitro cellular expression systems. Vascular injury and mismatched allograft rejection will be evaluated using mutant mice deficient in cd39 or animals with vascular upregulation of NTPDases achieved by either somatic recombination or transgenic approaches. These experiments should delineate involvement of NTPDases and P2signaling in inflammation associated with arterial/venous injury and also culminating in allograft failure.